The present invention relates to a method for the humanization of antibodies, by means of determining and comparing three-dimensional structures, humanized antibodies thereby obtained and their uses in therapy and diagnostics in vivo.
The therapeutic and diagnostic application of monoclonal antibodies of animal origins in humans has fundamental contraindications especially for therapeutic regimes which necessitate for repeated administrations. In particular, murine monoclonal antibodies have a relatively short half-life and, when used in humans, lack some fundamental functional characteristics of immunoglobulins, such as complement-dependent cytotoxicity and cell-mediated cytotoxicity.
Moreover, monoclonal antibodies of non-human origin contain immunogenic amino acid sequences if injected into patients. Numerous studies have shown that after the injection of an exogenous antibody, subjects develop a rather strong immune reaction against the antibody itself (known as HAMA—human anti-mouse antibodies—reaction), completely eliminating its therapeutic usefulness, with the formation of immunocomplexes, alteration of pharmacokinetics, production of allergic reactions, etc. Moreover, considering the growing number of different monoclonal antibodies developed in mice or in other mammals (and thus antigenic for humans) for the therapy of different pathologies, treatments, also for non correlated therapies can be ineffective or even dangerous due to cross-reactivity. Although the production of so-called chimeric antibodies (variable murine regions joined to constant regions of human origin) has yielded some positive result, a significant immunogenicity problem still remains.
Humanized antibodies have at least three potential advantages with respect to antibodies of animal origin in the field of therapeutic use in humans. In the first place, the effector region, being human, can better interact with the other parts of the human immune system, destroying target cells more efficiently by means of complement-dependent cytotoxicity, or cell-mediated, antibody dependent cytotoxicity. Moreover, the human immune system does not recognize the framework or the constant region (C) of the humanized antibody as exogenous, and hence the antibody response against the humanized antibody is minimized, both relative to that against a murine antibody (totally extraneous) and relative to the response induced by a chimeric antibody (partially extraneous).
It has been reported that murine antibodies injected into humans have a much shorter half-life time than normal antibodies (Shaw et al., 1987). Humanized antibodies have a very similar half life to that of natural human antibodies, allowing less frequent administration and lower doses.
The basic principle of humanization is configured in transferring the specificity of antigen recognition, i.e. the CDR domains, in the context of a human immunoglobulin (“CDR grafting”, Winter and Milstein, 1991). Several examples of humanized antibodies, produced in the attempt to solve the problem of immunogenicity, have been reported (Maeda et al., 1991; Singer et al., 1993; Tempest et al., 1994; Kettleborough et al., 1991; Hsiao et al., 1994; Baca et al., 1997; Leger et al., 1997; Ellis et al., 1995; Sato et al., 1994; Jones et al., 1986; Benhar et al., 1994; Sha and Xiang, 1994; Shearman et al., 1991; Rosok et al., 1996; Gussow & Seemann, 1991; Couto et al., 1994; Kashmiri et al., 1995; Baker et al., 1994; Riechmann et al., 1988; Gorman et al., 1991; Verhoeyen et al., 1988; Foote & Winter, 1992; Lewis & Crowe, 1991; Co et al., 1991; Co et al., 1991; Verhoeyen et al., 1991; Eigenbrot et al., 1994; Hamilton et al., 1997; Tempest et al., 1995; Verhoeyen et al., 1993; Cook et al., 1996; Poul et al., 1995; Co et al., 1992; Graziano et al., 1995; Presta et al., 1993; Hakimi et al., 1993; Roguska et al., 1996; Adair et al., 1994; Sato et al., 1993; Tempest et al., 1991; Sato et al., 1996; Kolbinger et al., 1993; Zhu and Carter, 1995; Sims et al., 1993; Routledge et al., 1991; Roguska et al., 1994; Queen et al., 1989; Carter et al., 1992).
The transcription of an antibody from animal (generally murine) to humanized entails the compromise between opposite requirements, whose solution varies case by case. To minimize immunogenicity, immunoglobulin shall maintain as much of the accepting human sequence as possible. In any case, to preserve the original binding properties, the immunoglobulin framework should contain a sufficient number of mutations in the accepting human sequence to guarantee that the conformation of the CDR regions is as similar as possible to that in the donor murine immunoglobulin. As a consequence of these opposite considerations, for many humanized antibodies a significant loss in binding affinity with respect to the corresponding murine antibodies has been reported (Jones et al., 1986; Shearman et al., 1991; Kettleborough, 1991; Gorman et al., 1991; Riechmann et al., 1988).
Currently, the most common method for the production of humanized immunoglobulin is based on the use of appropriate genomic, synthetic sequences, as well as cDNA (Reichmann et al., 1988).
The patent application EP 592106 discloses a method for the humanization of antibodies from rodents. The method is based on the identification of the amino acid residues exposed at the surface of the three-dimensional structure of the antibody to be humanized, on the identification of the amino acid residues in the same positions on the corresponding human antibody, and on the replacement of the residues identified in the sequence of the rodent antibody with those identified in the human antibody.